The Lac repressor provides a reversible gene expression system in undifferentiated and differentiated embryonic stem cell

Control of mammalian gene promoters by the bacterial LacI repressor provides reversible regulation and dose-response levels of derepressed expression by the lactose analog isopropyl thiogalactose (IPTG). Here, we show that insertion of LacI-binding sites in the ubiquitous β-actin promoter confers a strong and dose-dependent IPTG-regulatable expression of transiently transfected reporter genes in mouse embryonic stem (ES) cells expressing LacI. We established ES cell lines stably expressing reporter genes under inducible control and found a five- to tenfold IPTG induction of transgene expression. The kinetics of induction is rapid and stable, and can be rapidly reversed after IPTG removal. Importantly, this regulatable expression was maintained throughout the differentiation process of ES cells, and observed in individual differentiated cardiomyocyte-like cells and neuronal-like cells. This reversible system is the first to function from undifferentiated to individual welldifferentiated ES cells, providing a very useful tool to understand molecular mechanisms underlying ES cell self-renewal, commitment and differentiation.Received 17 March 2005; received after revision 19 April 2005; accepted 25 April 2005     

Crosstalk of protein kinase C ε with Smad2/3 promotes tumor cell proliferation in prostate cancer cells by enhancing aerobic glycolysis

Protein kinase C ε (PKCε) has emerged as an oncogenic protein kinase and plays important roles in cancer cell survival, proliferation, and invasion. It is, however, still unknown whether PKCε affects cell proliferation via glucose metabolism in cancer cells. Here we report a novel function of PKCε that provides growth advantages for cancer cells by enhancing tumor cells glycolysis. We found that either PKCε or Smad2/3 promoted aerobic glycolysis, expression of the glycolytic genes encoding HIF-1α, HKII, PFKP and MCT4, and tumor cell proliferation, while overexpression of PKCε or Smad3 enhanced aerobic glycolysis and cell proliferation in a protein kinase D- or TGF-β-independent manner in PC-3M and DU145 prostate cancer cells. The effects of PKCε silencing were reversed by ectopic expression of Smad3. PKCε or Smad3 ectopic expression-induced increase in cell growth was antagonized by inhibition of lactate transportation. Furthermore, interaction of endogenous PKCε with Smad2/3 was primarily responsible for phosphorylation of Ser213 in the Samd3 linker region, and resulted in Smad3 binding to the promoter of the glycolytic genes, thereby promoting cell proliferation. Forced expression of mutant Smad3 (S213A) attenuated PKCε-stimulated protein overexpression of the glycolytic genes. Thus, our results demonstrate a novel PKCε function that promotes cell growth in prostate cancer cells by increasing aerobic glycolysis through crosstalk between PKCε and Smad2/3.     

Silencing of RB1 but not of RB2/P130 induces cellular senescence and impairs the differentiation potential of human mesenchymal stem cells

Stem cell senescence is considered deleterious because it may impair tissue renewal and function. On the other hand, senescence may arrest the uncontrolled growth of transformed stem cells and protect organisms from cancer. This double function of senescence is strictly linked to the activity of genes that the control cell cycle such as the retinoblastoma proteins RB1, RB2/P130, and P107. We took advantage of the RNA interference technique to analyze the role of these proteins in the biology of mesenchymal stem cells (MSC). Cells lacking RB1 were prone to DNA damage. They showed elevated levels of p53 and p21^cip1 and increased regulation of RB2/P130 and P107 expression. These cells gradually adopted a senescent phenotype with impairment of self-renewal properties. No significant modification of cell growth was observed as it occurs in other cell types or systems. In cells with silenced RB2/P130, we detected a reduction of DNA damage along with a higher proliferation rate, an increase in clonogenic ability, and the diminution of apoptosis and senescence. Cells with silenced RB2/P130 were cultivated for extended periods of time without adopting a transformed phenotype. Of note, acute lowering of P107 did not induce relevant changes in the in vitro behavior of MSC. We also analyzed cell commitment and the osteo-chondro-adipogenic differentiation process of clones derived by MSC cultures. In all clones obtained from cells with silenced retinoblastoma genes, we observed a reduction in the ability to differentiate compared with the control clones. In summary, our data show evidence that the silencing of the expression of RB1 or RB2/P130 is not compensated by other gene family members, and this profoundly affects MSC functions.     

Connexin 43 a check-point component of cell proliferation implicated in a wide range of human testis diseases

Gap junction channels link cytoplasms of adjacent cells. Connexins, their constitutive proteins, are essential in cell homeostasis and are implicated in numerous physiological processes. Spermatogenesis is a sophisticated model of germ cell proliferation, differentiation, survival, and apoptosis, in which a connexin isotype, connexin 43, plays a crucial role as evidenced by genomic approaches based on gene deletion. The balance between cell proliferation/differentiation/apoptosis is a prerequisite for maintaining levels of spermatozoa essential for fertility and for limiting anarchic cell proliferation, a major risk of testis tumor. The present review highlights the emerging role of connexins in testis pathogenesis, focusing specifically on two intimately interconnected human testicular diseases (azoospermia with impaired spermatogenesis and testicular germ cell tumors), whose incidence increased during the last decades. This work proposes connexin 43 as a potential cancer diagnostic and prognostic marker, as well as a promising therapeutic target for testicular diseases.     

OX40 promotes obesity-induced adipose inflammation and insulin resistance

Adaptive immunity plays a critical role in IR and T2DM development; however, the biological mechanisms linking T cell costimulation and glucose metabolism have not been fully elucidated. In this study, we demonstrated that the costimulatory molecule OX40 controls T cell activation and IR development. Inflammatory cell accumulation and enhanced proinflammatory gene expression, as well as high OX40 expression levels on CD4⁺ T cells, were observed in the adipose tissues of mice with diet-induced obesity. OX40-KO mice exhibited significantly less weight gain and lower fasting glucose levels than those of WT mice, without obvious adipose tissue inflammation. The effects of OX40 on IR are mechanistically linked to the promotion of T cell activation, Th1 cell differentiation and proliferation—as well as the attenuation of Treg suppressive activity and the enhancement of proinflammatory cytokine production—in adipose tissues. Furthermore, OX40 expression on T cells was positively associated with obesity in humans, suggesting that our findings are clinically relevant. In summary, our study revealed that OX40 in CD4⁺ T cells is crucial for adipose tissue inflammation and IR development. Therefore, the OX40 signaling pathway may be a new target for preventing or treating obesity-related IR and T2DM.     

Crossing paths: interactions between the cell death machinery and growth factor survival signals

Cytokines and growth factors play a crucial role in the maintenance of haematopoietic homeostasis. They transduce signals that regulate the competing commitments of haematopoietic stem cells, quiescence or proliferation, retention of stem cell pluripotency or differentiation, and survival or demise. When the balance between these commitments and the requirements of the organisms is disturbed, particularly when it favours survival and proliferation, cancer may result. Cell death provoked by loss of growth factor signalling is regulated by the Bcl-2 family of apoptosis regulators, and thus survival messages transduced by growth factors must regulate the activity of these proteins. Many aspects of direct interactions between cytokine signalling and regulation of apoptosis remain elusive. In this review, we explore the mechanisms by which cytokines, in particular Interleukin-3 and granulocyte–macrophage colony-stimulating factor, promote cell survival and suppress apoptosis as models of how cytokine signalling and apoptotic pathways intersect.     

Hypoxia in the regulation of neural stem cells

In aerobic organisms, oxygen is a critical factor in tissue and organ morphogenesis from embryonic development throughout post-natal life, as it regulates various intracellular pathways involved in cellular metabolism, proliferation, survival and fate. In the mammalian central nervous system, oxygen plays a critical role in regulating the growth and differentiation state of neural stem cells (NSCs), multipotent neuronal precursor cells that reside in a particular microenvironment called the neural stem cell niche and that, under certain physiological and pathological conditions, differentiate into fully functional mature neurons, even in adults. In both experimental and clinical settings, oxygen is one of the main factors influencing NSCs. In particular, the physiological condition of mild hypoxia (2.5–5.0% O₂) typical of neural tissues promotes NSC self-renewal; it also favors the success of engraftment when in vitro-expanded NSCs are transplanted into brain of experimental animals. In this review, we analyze how O₂ and specifically hypoxia impact on NSC self-renewal, differentiation, maturation, and homing in various in vitro and in vivo settings, including cerebral ischemia, so as to define the O₂ conditions for successful cell replacement therapy in the treatment of brain injury and neurodegenerative diseases.     

Fluid shear stress-induced TGF-β/ALK5 signaling in renal epithelial cells is modulated by MEK1/2

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-β signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-β signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1 ^?/? cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-β-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-β signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-β/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.     

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNA^Met. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNA^Met methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.     

Divergent effects of the major mast cell products histamine, tryptase and TNF-alpha on human fibroblast behaviour

Fibroblast proliferation is a key process in tissue remodeling and mast cells (MCs) are thought to play a crucial role. Having established that the three major MC products, tryptase, histamine and TNF-alpha (TNF) are normally present in human skin MCs, which are in close proximity to dermal fibroblasts, we studied their individual effects on cell cycle-controlled human dermal fibroblasts (HFFF2). These cells express receptors (H1, PAR2, TNFR1/2) for the major MC mediators, but only tryptase or a PAR2 agonist peptide stimulated proliferation and gene expression. TNF was antimitotic, and histamine, while elevating intracellular Ca²⁺ levels at high concentrations, did not affect proliferation. We conclude that MC products but also composition and numbers of respective receptors on fibroblasts are crucially responsible for fibroproliferative events. Received: 28 June 2005; received after revision 28 September 2005; accepted 6 October 2005